The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled use and the remarkable ability of bacterial populations to develop resistance to these drugs has severely restricted the clinical usefulness of many antibiotics. We are now at a critical juncture where the majority of useful antibiotics have known and sometimes extensive resistance, and few novel replacements or strategies to combat the resistance problem exist. The aminoglycoside antibiotics are one important example of a large group of drugs with retained activity against key human pathogens but where the problem of resistance continues to increase. Of particular concern is the increasing identification over the last decade of pathogenic bacteria harboring exceptionally broad- spectrum and high-level aminoglycoside resistance determinants. These 16S ribosomal RNA (rRNA) methyltransferases chemically modify the aminoglycoside binding site on the 16S rRNA of the small (30S) bacterial ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S rRNA on nucleoside guanosine 1405 or adenosine 1408, to create m7G1405 and m1A1408 modifications, respectively. Functionally analogous self-protection resistance enzymes of both types were earlier identified in aminoglycoside-producing bacteria, and it is thought that these genes have laterally transferred to the pathogenic bacteria. Recent studies from our lab and others revealed the molecular structures of each enzyme, of both drug producer and pathogenic origin, and provided a first view of an m1A1408 methyltransferase bound to its substrate. However, additional evidence suggests that this enzyme family may employ diverse mechanisms to accomplish substrate specificity and modification. Further, comparatively little is known about how m7G1405 achieve the equivalent molecular functions. This proposal describes experiments in two connected aims that will directly address these deficiencies. In Aim 1 we will define the molecular mechanisms employed by the m1A1408 methyltransferases using a combination of biochemical, molecular biological and structural biology approaches to determine the molecular bases for control of m1A1408 methyltransferase activity by the S-adenosyl-L-methionine cosubstrate, and by the 30S subunit substrate. Aim 2 will provide the first detailed molecular insights into 30S recognition and modification by the m7G1405 methyltransferases using an innovative, interdisciplinary combination of approaches including synthetic chemistry to produce novel SAM analogs that will enable for the first time essential biochemical and structural studies of these clinical important resistance enzymes. Together these studies will provide novel and fundamentally important insights into the structures, activities and mechanisms of action of the aminoglycoside-resistance methyltransferases and will directly lay a secure foundation for future development of new strategies to counter the resistance they confer.